A human papillomavirus 16 E2-TopBP1 dependent SIRT1-p300 acetylation switch regulates mitotic viral and human protein levels and activates the DNA damage response

ABSTRACT An interaction between human papillomavirus 16 (HPV16) E2 and the cellular proteins TopBP1 and BRD4 is required for E2 plasmid segregation function. The E2-TopBP1 interaction promotes increased mitotic E2 protein levels in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes immortalized by HPV16 (HFK + HPV16). SIRT1 deacetylation reduces E2 protein stability and here we demonstrate that increased E2 acetylation occurs during mitosis in a TopBP1 interacting-dependent manner, promoting E2 mitotic stabilization. p300 mediates E2 acetylation and acetylation is increased due to E2 switching off SIRT1 function during mitosis in a TopBP1 interacting-dependent manner, confirmed by increased p53 stability and acetylation on lysine 382, a known target for SIRT1 deacetylation. SIRT1 can complex with E2 in growing cells but is unable to do so during mitosis due to the E2-TopBP1 interaction; SIRT1 is also unable to complex with p53 in mitotic E2 wild-type cells but can complex with p53 outside of mitosis. E2 lysines 111 and 112 are highly conserved residues across all E2 proteins and we demonstrate that K111 hyper-acetylation occurs during mitosis, promoting E2 interaction with Topoisomerase 1 (Top1). We demonstrate that K112 ubiquitination promotes E2 proteasomal degradation during mitosis. E2-TopBP1 interaction promotes mitotic acetylation of CHK2, promoting phosphorylation and activation of the DNA damage response (DDR). The results present a new model in which the E2-TopBP1 complex inactivates SIRT1 during mitosis, and activates the DDR. This is a novel mechanism of HPV16 activation of the DDR, a requirement for the viral life cycle. IMPORTANCE Human papillomaviruses (HPVs) are causative agents in around 5% of all human cancers. While there are prophylactic vaccines that will significantly alleviate HPV disease burden on future generations, there are currently no anti-viral strategies available for the treatment of HPV cancers. To generate such reagents, we must understand more about the HPV life cycle, and in particular about viral-host interactions. Here, we describe a novel mitotic complex generated by the HPV16 E2 protein interacting with the host protein TopBP1 that controls the function of the deacetylase SIRT1. The E2-TopBP1 interaction disrupts SIRT1 function during mitosis in order to enhance acetylation and stability of viral and host proteins. We also demonstrate that the E2-TopBP1 interaction activates the DDR. This novel complex is essential for the HPV16 life cycle and represents a novel anti-viral therapeutic target.

tightly regulated via an intricate interaction between viral and host factors, and cancer results when this regulation is disrupted, preventing differentiation, and promoting continued proliferation which results in the accumulation of host cell DNA damage (3).The viral oncogenes E6 and E7 both contribute to the regulation of cell differentiation and proliferation via interaction with host factors, including p53 and pRb, respectively (4).The viral genome is an 8kbp DNA episome whose replication is dependent upon the viral factors E1 and E2 interacting with host factors (5,6).
E2 has a carboxyl terminus dimerization and DNA binding domain, binding to 12 bp palindromic sequences in the viral genome that surround the A-T-rich viral origin of replication (5).Following binding, the E2 amino-terminal domain recruits the viral helicase E1 to the origin via a protein-protein interaction, whereupon E1 forms a di-hexameric helicase that activates viral replication in association with host polymerases (7,8).In addition to viral replication, the E2 protein can also regulate viral and host gene transcription via interaction with host proteins, including BRD4 (9)(10)(11)(12).A final role for E2 in the viral life cycle is segregation of the viral genome, where E2 acts as a "bridge" between the viral genome and host chromatin during mitosis to ensure the viral genome resides in daughter nuclei following cell division (13).Initial studies with BPV1 E2 demonstrated that the host protein BRD4 was required for E2 interaction with mitotic chromatin and some studies indicated that this was also the mitotic chromatin receptor for HPV16 E2, while others suggested an alternative host factor was responsible for HPV16 E2 interaction with host mitotic chromatin (14)(15)(16)(17)(18) We demonstrated that an interaction between HPV16 E2 (from now on, E2 will mean HPV16 E2 unless stated otherwise) and TopBP1 is essential for E2 interaction with mitotic chromatin and E2 plasmid segregation function, and that the E2-TopBP1 interaction is dependent upon E2 phosphorylation on serine 23 by CK2 (19)(20)(21).More recently, we demonstrated that an E2 interaction with both TopBP1 and BRD4 is required for E2 interaction with mitotic chromatin and E2 plasmid segregation function (22).During these studies, we observed a stabilization of E2 during mitosis that is dependent upon an interaction with TopBP1 but not BRD4.In addition, TopBP1 was also stabilized in an E2 interaction-dependent manner.These observations were made in the hTERT-immor talized human foreskin keratinocytes (HFKs) N/Tert-1 and U2OS cells, as well as in human foreskin keratinocytes immortalized by HPV16.
Previously, we demonstrated that the class III deacetylase SIRT1 can deacetylate E2, and that this deacetylation reduces E2 protein stability, while others have demonstrated a critical role for SIRT1 during the HPV31 life cycle (23,24).SIRT1 can also regulate the acetylation status and function of TopBP1 (25,26).Here, we demonstrate that, during mitosis in N/Tert-1 and U2OS cells, the interaction between E2 and SIRT1 is disrupted in an E2-TopBP1 interacting-dependent manner and that E2 acetylation is increased.E2 acetylation occurs on lysine 111 (K111) and this acetylation promotes interaction with topoisomerase 1 (Top1) and protects lysine 112 (K112) from ubiquitination.This explains why acetylation of K111 promotes E2 stabilization during mitosis.We demon strate aberrant interaction of the E2 K mutants with mitotic chromatin, and that the K111R mutant is defective in transient replication and transcription assays.Introduction of the E2 K mutants into the genome abrogated the immortalization potential of HPV16.SIRT1 is also inactive, in an E2-TopBP1 interacting-dependent manner, on p53 during mitosis resulting in enhanced p53 expression and acetylation.In HFK + HPV16 cells, SIRT1 expression is reduced at the protein level during mitosis and there is increased mitotic expression of E2 and p53 in these cells; a similar phenotype to that in the N/Tert-1 and U2OS cells expressing E2 only.Significantly, isogenic HFK immortalized by only E6 and E7 (HFK + E6/E7) do not show this phenotype; SIRT1 is unaffected and there is no increased acetylation and expression of p53 in these cells during mitosis.As well as p53, there is increased expression of CHK2 acetylation in E2-expressing cells due to the block in SIRT1 function.CHK2 acetylation promotes ATM phosphorylation (27) promoting activation of the DDR; we show there is a global increase in the DDR in mitotic E2-expressing cells that are dependent upon interaction with TopBP1.This is a completely novel mechanism for HPV16 activation of the DDR, a signaling pathway critical for HPV life cycles (28).While overexpression of E7 can induce DNA damage, there is almost no DDR activation in HFK cells immortalized by only the viral oncogenes E6 and E7.The results suggest that the E2-TopBP1 interaction is the major mechanism for HPV16 activation of the DDR.We also demonstrate that p300 is the acetylase responsible for the increased acetylation of viral and host proteins during mitosis in the HFK + HPV16 cells.The results demonstrate that an E2-TopBP1 interaction controls SIRT1 activity during mitosis and that this increases p300 acetylation of viral and cellular proteins and DDR activation, which is important during the HPV16 life cycle.

An E2-TopBP1 interaction turns off SIRT1 function during mitosis
Previously, we demonstrated increased expression of E2 and TopBP1 during mitosis in HFK + HPV16 cells, and that SIRT1 deacetylation of E2 reduces E2 protein expression (20,23).We therefore investigated whether the increased expression of E2 and TopBP1 during mitosis in HFK + HPV16 was due to increased E2 acetylation via disruption of SIRT1 mitotic function.Figure 1A demonstrates that SIRT1 protein expression is reduced in mitotic HFK + HPV16 cells (lane 4) but not in isogenic HFK immortalized by E6/E7 (lane 6), nor in N/Tert-1 cells that have been G418 selected (Vec, for Vector control, lane 2).The previously reported increase in E2 and TopBP1 was also detected in the HFK + HPV16 positive cells (lane 4).Mitotic cell enrichment was carried out by double thymidine blocking (DTB), followed by a release for 19 h (HFK) and 16 h (N/Tert-1 Vec), and mitotic enrichment is demonstrated by increased cyclin B expression (compare lanes 2, 4, and 6 with 1, 3, and 5).For the sake of simplicity, we will call these mitotically enriched cells mitotic, acknowledging that this is not a pure mitotic cell population.We did not want to use cell cycle inhibitors such as nocodazole as they are DNA-damaging agents and may disrupt TopBP1 function.In mitotic N/Tert-1 and HFK + E6/E7 cells, there is an increase in SIRT1 during mitosis (lanes 2 and 6) in sharp contrast to the reduction observed in HFK + HPV16 cells (lane 4), and no increase in TopBP1 expression.As TopBP1 is a DDR factor and HPVs activate the DDR to promote their life cycle, we looked at the levels of CHK2 in the mitotic cells.In HFK + HPV16 cells, there is increased expression of CHK2 in mitosis when compared with the two control lines (compare lane 4 with lanes 2 and 6).Using a pCHK2-specific antibody that recognizes ATM phosphorylated CHK2, we demonstrate an enhanced activation of CHK2 during mitosis in the HFK + HPV16 cells, but not in the N/Tert-1 + Vec or HFK + E6/E7 cells.An acetyl lysine co-immunoprecipitation (IP) determined that the acetylation of E2, TopBP1 CHK2, and p53 was increased during mitosis (Fig. 1B).Lane 1 is a co-IP with the control HA antibody, and it does not interact with any of the proteins under study.In N/Tert-1 and HFK + HPV16 cells, p53 is acetylated at time 0 h (0 hours) (lanes 2 and 4) and this acetylation is increased in mitotic HFK + HPV16 cells (lane 5) and not in N/Tert-1 cells (lane 3), where p53 expression is reduced (Fig. 1A).p53 levels in HFK + HPV16 cells are detectable as E6 is spliced to E6* which removes the p53 degradation domain of E6 (29).We have demonstrated abundant p53 expression in HPV-positive head and neck cancer cell lines and patient-derived xenografts, as well as in primary keratinocytes immortalized by the entire HPV16 genome (30).No p53 is detectable in the HFK + E6/E7 cells as E6 is not spliced to E6* and can therefore target p53 for proteasomal degradation.The acetylation of E2 is detectable in 0 h HFK + HPV16 cells and this increases during mitosis (lanes 4 and 5), reflective of the increased E2 protein expression (Fig. 1A).Acetylation of CHK2 has been shown to activate ATM phosphorylation and promote the DDR (27), and CHK2 is selectively acetylated in mitosis in the HFK + HPV16 cells.There is no detectable acetylation of the residual levels of SIRT1 in mitotic HFK + HPV16 (lane 5).Next, we investigated whether the residual SIRT1 in mitotic cells was able to interact with TopBP1, E2, CHK2, and p53 using a SIRT1 co-immunoprecipitation (Fig. 1C).TopBP1 interaction with SIRT1 is not disrupted in mitosis in any of the samples.In HFK + HPV16 cells, SIRT1 interaction with E2, CHK2, and p53 is disrupted during mitosis (lane 5). Figure S1 quantitates repeats of  One of the functions of SIRT1 during mitosis is to deacetylate histones and promote chromatin condensation (31,32).The reduction in SIRT1 function during mitosis could therefore alter transcription from the viral and host genome in HFK + HPV16 cells.Figure 1D demonstrates that there is a significant increase in E2 and p53 RNA levels in the mitotically enriched HFK + HPV16 cells; p53 RNA levels are not increased in mitosis in either N/Tert-1 Vec or HFK + E6/E7 cells.With SIRT1 there is a reduction in mitotic HFK + HPV16 cells and an increase in HFK + E6/E7, although neither reached significance.When paired, there is a significant difference between mitotic SIRT1 RNA levels in HFK + HPV16 cells versus HFK + E6/E7 cells.There is no significant change in the SIRT1 levels in N/Tert-1 Vec cells.Compared to the levels of protein changes observed (Fig. 1A), the changes in RNA levels were minimal although may contribute to the changes in protein levels observed in mitosis (Fig. 1A; Fig. S1).There is no significant change in TopBP1 or CHK2 RNA levels in any of the samples.
As the changes in p53 and SIRT1 levels were not observed in HFK + E6/E7 (Fig. 1), and E2 increases TopBP1 protein levels during mitosis (20,21), the ability of E2 to regulate SIRT1 function during mitosis via interaction with TopBP1 was investigated in N/Tert-1 and U2OS cells (Fig. 2). Figure 2A demonstrates that in N/Tert-1 cells stably expressing E2-WT, there is an increase in TopBP1, E2, CHK2, and p53 during mitosis, which is similar to the HFK + HPV16 cells (Fig. 1A).An E2 mutant that cannot bind TopBP1 (E2-S23A), did not increase the expression of TopBP1, p53, CHK2, or E2 during mitosis (compare lane 6 with lane 4).Ac-p53 (K382) expression was increased in mitosis in the E2-WT cells; this lysine is targeted by SIRT1 and confirms SIRT1 inactivation during mitosis.These data were generated from two different gels, the separation of which is indicated by the lane number position.An acetyl lysine immunoprecipitation demonstrated increased acetylation of TopBP1, CHK2 and E2 during mitosis, and a slight increase in acetylated p53 (compare lane 5 with lanes 3 and 7).In both N/Tert-1 Vec and E2-S23A cells, there is a reduction in p53 protein levels during mitosis (Fig. 2A).Unlike HFK + HPV16 cells, E2-WT did not reduce SIRT1 protein levels in mitosis when compared with non-mitotic cells (Fig. 2A, lanes 3 and 4), although SIRT1 protein levels did not increase as they do in Vec and E2-S23A cells (lanes 2 and 6, Fig. 2A).However, as in HFK + HPV16 cells, there is a reduction in SIRT1 acetylation levels during mitosis only in the presence of E2-WT (Fig. 2B, lane 5). Figure 2C demonstrates that the ability of SIRT1 to complex with p53, CHK2, and E2 during mitosis is abrogated only in E2-WT cells, as it is in HFK + HPV16 cells.Figure S2 quantitates repeats of Fig. 2A demonstrating significant changes in TopBP1, SIRT1, E2, and p53 between the samples.
The results in Fig. 2 demonstrate that E2 manipulates SIRT1 function during mitosis in a TopBP1 interaction-dependent manner, and Fig. 1 confirms this happens in HFK + HPV16 cells.E2 interaction with TopBP1 is required for increased p53 acetylation on lysine 382, an acetylation known to stabilize p53.We next determined the E2 residues that regulate E2 acetylation and stability during mitosis.

E2 K111 and K112 regulate the stability of E2 during mitosis
E2 lysines 111 and 112 (K111 and K112) are highly conserved across HPV types, and HPV16 E2 K111 is acetylated (33).A panel of N/Tert-1 cells was established stably expressing K111 and K112 E2 mutants that mimicked acetylation (K to Q) or mimicked non-acetylation (K to R) (Fig. 3A).Using DTB and release for 16 h, mitotic enrichment was carried out and western blotting demonstrated the expression of the E2 proteins and cyclin B expression demonstrated mitotic enrichment (Fig. 3B).E2-WT had increased expression during mitosis (lanes 4 and 12), E2-K111R (mimicking non-acetylation) had no  detectable expression during mitosis (lane 6), while E2-K111Q (mimicking acetylation) expressed similarly to E2-WT during mitosis (compare lane 14 with lane 12).E2-K112R levels increased during mitosis similarly to E2-WT (compare lane 8 with lane 4).E2-K111R + K112R (double lysine mutant) protein levels did not increase during mitosis but were detectable.All of the mutants were able to complex with TopBP1, if they were detectable in the input blot (Fig. 3C).An acetyl-lysine immunoprecipitation demonstrated increased acetylation of E2-WT and E2-K112R during mitosis, none of the other mutants were pulled down by the acetyl-lysine antibody, including K111R in non-mitotic cells (lane 6) (Fig. 3D).The results demonstrate that the acetyl-lysine antibody used in these experi ments detects one major site of acetylation on E2 in and out of mitosis, K111.It is possible that additional E2 residues can be acetylated but are not recognized by the antibody used.Acetylation of K111 promotes interaction with Topoisomerase 1 (Top1) (33,34) and we investigated the ability of the E2 mutants to interact with Top1 (Fig. 3E).E2-K111R and E2-K111R + K112R were unable to complex with Top1 (lanes 6 and 7, and 17 and 18, respectively).The significant mitotic increase of E2-WT, K112R, and K111Q protein expression, as well as TopBP1 in these three samples, when compared with the time 0 h sample (Fig. 3B) was confirmed using multiple sample blots (Fig. S6).E2 nor TopBP1 RNA levels were changed in mitosis in any of the cell lines, nor were there any changes in expression due to the E2 mutations between samples (Fig. S7).
The recovery of E2K111R + K112R protein expression in mitosis, when compared with E2-K111R, suggested that K112 was ubiquitinated, targeting the E2 protein for proteasomal degradation.To test this, the proteasomal inhibitor MG132 was added 15 h following release from the DTB and E2 protein levels determined at 16 and 18 h following DTB release (Fig. 3F).Without addition of MG132, E2-K111R was undetectable during mitosis, similar to Fig. 3B.However, the addition of MG132 for 1 h prior to cell harvesting resulted in the detection of E2-K111R in mitotic cells, demonstrating that proteasomal inhibition restored expression (lane 11).Interestingly, E2-WT levels 3 h following the addition of MG132 are reduced, suggesting the decrease of E2 protein levels following exit from mitosis may not be mediated by proteasomal degradation.To determine whether E2-K111R has increased ubiquitination in mitotic cells, we used the Chromo Tek Ubiquitin-Trap (Fig. 3G).The samples assayed were mitotic samples treated with and without MG132 for 1 h prior to protein extraction (Fig. 3F).Lane 7 demonstrates that in mitotic cells treated with MG132 E2-K111R is ubiquitinated, which would target the protein for proteasomal turnover, explaining the lack of E2-K111R protein expression during mitosis.The results from Fig. 1-3 support a model in which SIRT1 function is, at least partially, inactivated during mitosis in an E2-TopBP1 interacting-dependent manner.This SIRT1 inactivation results in enhanced acetylation of E2 K111, promoting interac tion with Top1.This interaction protects E2 K112 from ubiquitination and proteasomal turnover during mitosis promoting increased E2 protein levels.

Lysine mutations in E2 disrupt E2 mitotic interaction and abolish HPV16 primary cell immortalization
To determine the cellular localization of the E2 lysine mutants, stable U2OS cells expressing the mutants were generated (Fig. 4A).Increased E2 expression and acety lation occur in U2OS cells in an identical manner to that in N/Tert-1 cells (Fig. 2), and imaging mitotic U2OS cells is more straightforward than imaging mitotic N/Tert-1 cells.Fig. 4B represents examples of mitotic U2OS cells stably expressing the indica ted mutants.In the absence of E2, TopBP1 is located on mitotic chromatin (Vec, top panels) and WT-E2 is co-located with TopBP1 on mitotic chromatin (second panel down), as previously reported (20).E2-K111R expression during mitosis is not detected by western blotting and in immunofluorescence there was a reduced E2-K111R signal in mitotic cells (third panel down).The interaction of TopBP1 with mitotic chromatin is not affected by E2-K111R expression and the E2-K111R is not detected on the mitotic chromatin.E2-K111Q is located on mitotic chromatin (fourth panel down), but this mutant removes TopBP1.E2 K112R (fifth panel down), well expressed during mitosis, has a reduced location to mitotic chromatin when compared with E2-WT and does not disrupt the ability of TopBP1 mitotic interaction.E2-K111R + K112R has a similar phenotype as E2-K111R (sixth panel down).In interphase cells, E2-K111R was detected in the cytoplasm when compared with E2-WT, and E2-K112R and E2-K111R + K112R also had more cytoplasmic E2 staining.The levels of E2 and TopBP1 interaction with mitotic chromatin were determined using the Keyence imaging system over multiple mitotic cells and confirmed the phenotypes observed in Fig. 4B (Fig. 4C).Nuclear and cytoplas mic E2 staining was also quantitated and confirmed significantly more cytoplasmic staining for E2-K111R, E2-K112R, and E2-K111R + K112R (Fig. 4D).Previous studies with cottontail rabbit papillomavirus E2 mutations in these residues transiently overexpressed in C33a cells also identified cytoplasmic localization of K111 mutants (35).
TopBP1 is an active protein during mitosis and is critical for maintaining genome integrity (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46).The ability of E2-K111Q to remove TopBP1 from mitotic chromatin indicated this protein may disrupt mitosis.We investigated the number of mitotic cells that contained anaphase bridges and observed an increased number of cells with this phenotype in the E2-K111Q cells, when compared with control and other E2-expressing cells.Fig. 4E provides an enhanced image of one of the cells containing an anaphase bridge, and Fig. 4F provides a quantitation of the occurrence, demonstrating a significant increase in E2-K111Q cells.
The ability of the E2 K mutants to immortalize HFKs was investigated.HPV16 genomes were generated with E2 mutations encoding K111R, K111Q, and K112R, and along with wild-type HPV16 genomes were transfected into two independent primary foreskin keratinocyte donor cells.In both donors, HPV16 wild type generated immor talized clones that were grown out into cell lines; none of the K mutants were able to generate clones or immortalized lines (Table 1).This demonstrates that wild-type E2-K111 and K112 are critical for the immortalization of HFK.Androphy et al. demon strated that K111 is critical for the transcription and replication function of E2 and we observed the same phenotype (Fig. S8), all mutants except for K111R and K111R + K112R were similar to wild type in their transcription and replication function in C33a cells.The K111R phenotype could be related to a failure to complex with Top1 [Fig.3E and (33)], but could also be due to other reasons such as the failure to be expressed during mitosis that we observe here (Fig. 3).

p300 is the predominant mitotic acetylase for E2
p300 is a histone acetyltransferase involved in the acetylation and functional regula tion of E2 proteins (33,34,(47)(48)(49)(50)(51)(52).Knockdown of p300 in N/Tert-1 cells abolished the increase of E2 and TopBP1 mitotic protein levels (Fig. 5A, compare lane 4 with lane 8).An acetyl lysine immunoprecipitation demonstrated that p300 is required for detectable E2 acetylation in and out of mitosis (Fig. 5B, compare lanes 4 and 5 with 8 and 9).For TopBP1, there is a pull-down in all lanes demonstrating that p300 is not the only acetylase that can target TopBP1, although the increase in mitosis in the E2-expressing cells is mediated by p300 (compare lane 5 with 9).There is increased SIRT1 acetylation during mitosis in the E2-TopBP1 samples following p300 knockdown (Fig. 5B, compare lanes 5 and 9).This demonstrates that the E2-TopBP1 stabilization due to p300 acetylation prevents SIRT1 acetylation by another acetylase, and that this control is lost following p300 knockdown due to the reduction in E2-TopBP1 protein levels.p300 knockdown does not alter SIRT1 acetylation levels in N/Tert-1 Vec control cells demonstrating that p300 is not a SIRT1 acetylase.p300 is also acetylated but not altered by E2 expression or cell cycle changes (lanes 2-5).These experiments were repeated with an additional p300 siRNA and generated essentially the same results (Fig. S9). a Two independent human foreskin donors were transfected with the indicated HPV16 genomes and the subsequent growth of colonies and immortalized cell lines determined.K111R = E2 lysine 111 mutated to arginine; K111Q = E2 lysine 111 mutated to glutamine; K112R = E2 K112 mutated to arginine.
Next, we investigated whether p300 was responsible for E2 mitotic acetylation in HFK + HPV16 cells (Fig. 6A).Following p300 knockdown the increase in E2, TopBP1, CHK2, and p53 levels during mitosis is abolished (compare lane 4 with lane 8); p300 is a known p53 and CHK2 acetylase (27,53).An acetyl lysine IP (Fig. 6B) demonstrated that acetylation of E2, TopBP1, CHK2, and p53 was reduced following p300 knockdown, and that p300 is not the SIRT1 acetylase.We repeated these experiments with an additional p300 siRNA and got essentially identical results (Fig. S10).Overall, the results mimic those observed in the N/Tert-1 + E2 cells, including the regulation of SIRT1 acetylation (Fig. 5).We confirmed that E2 and TopBP1 co-localize on mitotic chromatin in HFK + HPV16 cells, although a significant proportion of TopBP1 does not locate to the mitotic chromatin, which is distinct from the N/Tert-1 cells (Fig. 6C).Finally, we investigated whether the E2-TopBP1 interaction activates a universal DDR during mitosis.To do this, extracts were western blotted with a pSQ antibody that recognizes phosphorylated serines before a glutamine, a substrate for ATM and ATR kinases.Figure 7A demonstrates that in HFK + HPV16 cells there is a strong mitotic activation of the DDR (lane 4); the top panel is a pSQ western blot and molecular weight markers are provided (in kD).The GAPDH served as a loading control and was carried out on the stripped blot.With N/Tert-1 and HFK + E6/E7, there is minimal pSQ detected during mitosis (lanes 2 and 6, respectively).At the 0 h time point, there is also more pSQ signal detected in the HFK + HPV16 cells compared to the others (lane 3 vs lanes 1 and 5).To determine whether this mitotic activation of the DDR is due to the E2-TopBP1 interaction, we carried out the same experiment with N/Tert-1+Vec, E2-WT, and E2-S23A (Fig. 7B).There is an activation of the DDR in mitotic E2-WT cells (lane 4) and much less so in Vec and E2-S23A cells (lanes 2 and 6, respectively).Overall, Fig. 7 demonstrates that the E2-TopBP1 interaction activates the DDR in mitosis during the HPV16 life cycle.

DISCUSSION
This report describes a novel E2-TopBP1 centered mitotic complex (ECMC) that controls mitotic acetylation and expression of proteins critical for the viral life cycle, including E2 and TopBP1.The increased acetylation is due to a reduction in SIRT1 function during mitosis in an E2-TopBP1-dependent manner.In HFK + HPV16 cells, there is a reduction of SIRT1 levels during mitosis; while in N/Tert-1 cells, SIRT1 function is turned off as demonstrated by increased acetylation of p53 on lysine 382, a known SIRT1 target residue.SIRT1 turn-off is dependent upon E2 interaction with TopBP1 as an E2 mutant that fails to bind TopBP1 (where serine 23 is changed to an alanine, S23A [21]) is unable to alter p53, TopBP1 or E2 acetylation during mitosis in N/Tert-1 or U2OS cells.During mitosis, SIRT1 is unable to complex with E2 or p53 (which it can outside of mitosis) suggesting that structural changes in the E2-TopBP1 complex during mitosis control SIRT1 partner protein interactions.TopBP1 remains complex with SIRT1 throughout mitosis, and SIRT1 can regulate the function of TopBP1 in metabolic and DNA damage pathways (25,54).Increased acetylation of TopBP1 occurs following activation of the DNA damage response (DDR) which inactivates SIRT1 function (25); this also increases p53 acetylation, stability, and function to mediate the DDR (55).The DDR is active in HPV-positive cells and is critical for the viral life cycle, as is the expression of SIRT1 (24,28,(56)(57)(58).Other SIRT1 substrates, including NBS1 and WRN, are critical for HPV life cycles (57,(59)(60)(61).During mitosis, SIRT1 condenses chromosomes due to deacetyla tion of histones (32), and the results presented here suggest that in HPV-transformed cells, there may be alternative mechanisms for carrying out this process as SIRT1 is, at least partially, inactivated.To our knowledge, we report for the first time that SIRT1 is acetylated and in E2-expressing cells this acetylation is reduced during mitosis.How the E2-TopBP1 interaction is regulating SIRT1 mitotic acetylation and function remains to be determined, but the altered SIRT1 acetylation likely contributes to the alteration in mitotic SIRT1 interacting partners and function.It is possible that mitotic kinases target the E2-TopBP1 complex to change the structure allowing for the sequestration of SIRT1 from binding partners and the blocking of its acetylation.The acetylation of SIRT1, unlike that of E2 and TopBP1, is independent of p300 and the SIRT1 acetylase could be targeted by mitotic kinases to block SIRT1 acetylation and therefore function.Future studies will focus on identifying the E2-TopBP1-dependent mechanism that regulates SIRT1 function during mitosis.
The results present a mechanism for regulating E2 stability during mitosis (Fig. 7).As cells enter mitosis, the ability of SIRT1 to bind and deacetylate E2 is abrogated resulting in increased K111 acetylation by p300.This promotes the recruitment of Top1 that prevents the ubiquitination of K112 resulting in enhanced E2 protein stability and expression.TopBP1 is a known interactor with ubiquitin ligases that could also be in the E2-TopBP1 complex, or could be independent of the complex (indicated by the question mark in the top panel of Fig. 8) (62).However, when K111 is mutated (K111R), there is a failure to interact with Top1 allowing ubiquitination of K112 and proteasomal degradation (Fig. 8, bottom panel).Undoubtedly, other protein factors are involved in the E2-TopBP1 complex and future studies will seek to characterize the E2-TopBP1 complex in and out of mitosis.
Another important aspect of the E2-TopBP1 interaction is activation of the DDR during mitosis.DDR activation is required for HPV life cycles, therefore its activation is critical (28).This is evidenced by increased acetylation and ATM phosphorylation of CHK2 (Fig. 1 and 2), and a global increase of pS-T/Q signal (Fig. 7).It is striking that there is no increase in the DDR in HFK + E6/E7 cells and the results suggest that the E2-TopBP1 interaction is the main driver of DDR activation during the HPV16 life cycle.E7 clearly plays a role in inducing damage to the host DNA (63), but the evidence presented here suggests it is not critical for DDR activation, but may amplify the DDR signaling following E2-TopBP1 activation.
We recently demonstrated that the E2-TopBP1 interaction is critical for the plasmid segregation function of E2 (20)(21)(22).This function of E2 promotes the nuclear localization of viral genomes following mitosis by allowing the viral genomes to "hitchhike" onto the host mitotic chromatin using the E2-TopBP1 interaction.Disruption of the E2-TopBP1 interaction also resulted in a loss of E2 protein expression in HFK + HPV16 cells, promot ing viral genome integration (19).In this report, we demonstrate that mutation of E2 K111 or K112 prevented the viral genome from immortalizing HFKs (Table 1).This failure could be due to the aberrant expression and mitotic interaction of the E2 mutants demonstrated in this report, resulting in a loss of the viral DNA from daughter nuclei following mitosis, reducing viral nuclear DNA to a level that does not support immortali zation.It is interesting that E2-K111Q can complex with mitotic chromatin and remove TopBP1.What could be occurring is that TopBP1 recruits E2-K111Q which then perma nently binds to another protein (BRD4 is an obvious candidate) changing TopBP1 structure (or BRD4 structure) to remove TopBP1 from the mitotic chromatin.
Increased p53 acetylation due to inactivation of SIRT1 following activation of the DDR ordinarily promotes the ability of p53 to activate genes that arrest the cell cycle and promote DNA repair (55).Therefore, the p53 acetylation during mitosis in HFK + HPV16 cells is surprising as these cells are proliferating and must pass through mitosis to allow continued proliferation.The role of p53 in the HPV16 life cycle is not clear.E6 expression certainly degrades p53 but in cells immortalized by the full genome E6 can be spliced to E6* which removes the ability to degrade p53 (29).Recently, we demonstrated robust p53 protein expression in HFK + HPV16, in HPV16-positive head and neck cancer cell lines, and in HPV16, positive head and neck cancer patient-derived xenografts (30).Overexpression of full-length E6 in HFK + HPV16 cells abrogated their growth (an E6 mutant unable to degrade p53 did not) suggesting that p53 may be important for the HPV16 life cycle.We have been unable to use CRISPR/Cas9 to remove p53 expression from HFK + HPV16 cells, even with target RNAs that do reduce p53 expression in non-HPV16 cells (not shown).Future studies will further investigate a possible role of p53 in the viral life cycle.p53 can directly interact with E2 and can regulate E2 replication function; therefore, it is possible that p53 plays a role in the regulation of viral replication (64,65).
Overall, the results identify a novel mitotic complex that is regulated by E2 interaction with TopBP1, which inhibits SIRT1 mitotic function, promoting acetylation of viral and host proteins.This interaction also activates the DDR during mitosis.We have already demonstrated that the E2-TopBP1 interaction is critical for the HPV16 life cycle, and the results here demonstrate that regulation of E2 K111 acetylation and K112 ubiquitination are also critical for HPV16 immortalization.Future studies will focus on gaining a greater understanding of this E2-TopBP1 centered mitotic complex and what the critical aspects are for the HPV16 life cycle.

Immortalization of human foreskin keratinocytes
HFKs were immortalized with HPV16 as described previously (21).Briefly, 2 × 10 5 low passage (p2-p5) primary cells were plated onto collagen-coated 6 cm dishes and, once at 60% confluency, were transfected with re-circularized HPV16 wild-type or mutant genomes alongside neomycin resistance plasmid, pCDNA3.1.These then underwent selection, consisting of 8 days of alternate treatment with media containing G418 and 10 µM Y-27632, and with mitomycin-c inactivated J2 fibroblasts.Once selected, cells were cultured in kSFM without additional drugs with mitomycin-c inactivated J2 fibroblasts, and transferred to 10 cm dishes.Once colonies were visible in the wild-type cells, media were removed and cells were washed two times with phosphate-buffered saline (PBS).About 5 mL of 0.5% crystal violet was added per dish, and then incubated shaking at room temperature for 30 min.Dishes were washed five times with PBS and plates were left to dry.Crystal violet images were scanned using the Odyssey CLx Imaging System and quantified by eye.Immortalization was done in duplicate in two donor backgrounds, individually.
HFKs were immortalized with HPV16 (WT) or immortalized with E6/E7 was cultured in Dermalife-K complete medium (Lifeline Cell Technology) as previously described (21).Mitomycin C-treated 3T3-J2 fibroblast feeders were plated 24 h prior to plating N/Tert-1-Vec or HFK cells on top of the feeders, in their respective cell culture medium and allowed to grow to 70% confluency.C33a cells were obtained from ATCC (ATCC HTB-31) and grown in DMEM supplemented with 10% FBS.In all cases, cell identity was confirmed via "fingerprinting, " and cell cultures were routinely tested for mycoplasma.

Cell synchronization
N/Tert-1 vector control cells (in K-SFM medium) and HFK cells immortalized with HPV16 genomes (HFK + HPV16) as well as HFK + E6/E7 cells (in Dermalife-K complete medium) were cultured with Mitomycin C-treated 3T3-J2 fibroblasts.Cells were plated at 5 × 10 5 density onto 100 mm plates.The cells were treated with 2 mM thymidine diluted in their respective medium for 16 h.Cells were then washed two times with PBS and recovered in their respective medium.After 8 h, to block the cells at G1/S phase, a second dose of 2 mM thymidine was added, and the cells were incubated for 17 h.The cells were then washed two times with PBS and recovered as before at the following time points.For HFK, cells were harvested at 0 h (G1/S phase) and 19 h (M1 phase).The above procedure was repeated in N/Tert-1 cells and U2OS cells expressing stable E2-WT, E2-S23A, E2-K111R, E2-K112R, E2-K111Q, E2-K111R + K112R, along with plasmid control cells which were plated at a density of 5 × 10 5 in K-SFM medium (for N/Tert-1 cells) or 3 × 10 5 in DMEM medium (for U2OS cells) on 100 mm plates and the double thymidine blocked N/Tert-1 cells were harvested at 0 h (G1/S phase) and 16 h (M1 phase).Double thymidine-blocked U2OS cells were harvested at 0 h (G1/S phase) and 8 h (M1 phase).Using the harvested cells at the time points mentioned, cell lysates were prepared, and immunoblotting was carried out.Cyclin B1 antibody was used to confirm the mitosis phase in these cells using immunoblotting as described below.

Immunoprecipitation
Protein lysate (250 µg) from indicated cells (prepared as described above) was incubated with primary antibody of interest or a HA tag antibody (used as a negative control). 1 µg of antibody per 100 µg protein lysate was used per reaction.The protein lysate-antibody mixture was made up to a total volume of 500 µL with lysis buffer supplemented with protease inhibitors and phosphatase inhibitor cocktail and rotated end-to-end at 4°C overnight.The next day, 40 µL of prewashed protein A beads per sample (Sigma; prewashed in lysis buffer as mentioned in the manufacturer's protocol) was added to the lysate-antibody mixture and rotated for an additional 4 h at 4°C.The samples were washed gently with 500 µL lysis buffer and centrifuged at 1,000 rcf for 3 min.This wash step was repeated three times.The bead pellet was resuspended in 4× Laemmli sample buffer (Bio-Rad), heat denatured, and centrifuged at 1,000 rcf for 3 min.Proteins were separated using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system and transferred onto a nitrocellulose membrane before probing for the presence of E2, TopBP1, p300, SIRT1, Top1, and p53 as per the western blotting protocol described above.

RNA isolation and SYBR green reverse transcription
RNA was isolated using the SV Total RNA isolation system (Promega) following the manufacturer's instructions.We reverse transcribed 2 µg of RNA into cDNA using the high-capacity reverse transcription kit (Applied Biosystems).cDNA was then processed for qPCR.

MG132 proteasomal inhibitor treatment
N/Tert-1 cells expressing stable E2-WT or E2-K111R, E2-1112R, E2-111Q, and E2-K111R + K1112R mutants were plated in KSFM media.Next, double thymidine treatment was carried out as described above.At 15 h post second thymidine recovery, cells were treated with 10 µM of MG132 (Z-Leu-Leu-Leu-al; Sigma, catalog no.C2211).Cells were then harvested at 1 h after MG132 treatment (corresponds to 16 h post second thymidine recovery; peak M1 phase in N/Tert-1 cells) and at 2 h after MG132 treatment (corresponds to 18 h post second thymidine recovery).Immunoblotting was carried out to detect E2 and TopBP1 levels.

Ubiquitin trap
Protein lysates from the above-mentioned cell synchronized and MGM132 treated N/ Tert-1 cells were harvested using NP40 protein lysis buffer.300 µg of protein lysate was incubated with 25 µL ChromoTek Ubiquitin-Trap Agarose slurry (Proteintech) equilibra ted in NP40 protein lysis buffer and placed at 4°C for 1 h with continual end-to-end rotation.The protein-bound Ubiquitin-Trap Agarose was washed three times in the NP40 lysis buffer by centrifugation at 1,000 rcf for 3 min and resuspended in 4× Laemmli sample buffer (Bio-Rad), heat denatured, and centrifuged at 1,000 rcf for 3 min.The supernatant was gel electrophoresed using an SDS-PAGE system which was later transferred onto a nitrocellulose membrane using the wet-blot transfer method.The membrane was probed with an E2 antibody to detect ubiquitinated E2.

Small interfering RNA treatment
HFK cells or N/Tert-1 cells were plated on top of Mitomycin C-treated 3T3-J2 fibroblast feeders in 100 mm plates in their respective media.The next day, cells were transfected with 10 µM of the small interfering RNA (siRNA) mentioned below.10 µM of a "non-tar geting" control, MISSION siRNA Universal Negative Control (Sigma-Aldrich; catalog no.SIC001), was used in our experiments.siRNA knockdown was carried out following the protocol from Lipofectamine RNAiMAX transfection (Invitrogen, catalog no.13778-100).Cells were harvested 48 h post-transfection and using the protocol as described above, immunoblotting for the protein of interest was done to confirm the knockdown.All siRNAs were purchased from Sigma-Aldrich: siRNA p300-A, 5′-TTGGACTACCCTATCAAGTA A-3′; siRNA p300-B, 5′-GGACUACCCUAUCAAGUAA-3′.

DNA replication assay
On 100 mm dishes, C33a cells were plated at 5 × 10 5 in DMEM + 10% FBS.The following day, plasmid DNA was transfected using the calcium phosphate method (74).Three days post-transfection, low molecular weight DNA was extracted using the Hirt method as previously described (75).The sample was extracted two times with phenol:chloro form:isoamyl alcohol (25:24:1) and precipitated with ethanol.Following centrifugation, the DNA pellet was washed with 70% ethanol, dried and resuspended in a total of 150 µL water.About 42 µL of the sample was digested with DpnI (New England Biolabs) overnight to remove unreplicated pOri16LacZ; the sample was then digested with ExoIII (New England Biolabs) for 1 h.Replication was determined by real-time PCR, as described previously (72).

Transcription assay
C33a cells were plated at 5 × 10 5 on 100 mm plates in DMEM medium with 10% FBS.The next day, the cells were transfected with either 1 µg HPV16 E2 plasmids (WT or E2-K mutants) and 1 µg ptk6E2-luc or 1 µg ptk6E2-luc alone using calcium phosphate transfection method.Briefly, the cells were harvested 72 h post-transfection utilizing the Promega reporter lysis buffer and analyzed for luciferase using the Promega luciferase assay system (catalog no.E1500).Concentrations were normalized to protein levels, as measured by the Bio-Rad protein estimation assay mentioned above.Relative fluorescence units were measured using the BioTek Synergy H1 hybrid reader.

Statistical analysis
All the experiments were carried out in triplicates in each of the mentioned cell lines and quantitation of the results represented as mean ± standard error (SE).Significance was determined using Student's t test.

FIG 1 (
FIG 1 (A) Increased expression of E2, TopBP1, and p53 in mitotic enriched HFK + HPV16 cells.A. N/Tert-1 (lanes 1 and 2), HFK + HPV16 (lanes 3 and 4), and HFK + E6/E7 (lanes 5 and 6) were arrested by double thymidine block (DTB) (lanes 1, 3, and 5) and released for 16 h (N/Tert1, lane 2) or 19 h (HFK + HPV16 and HFK + E6/E7, lanes 4 and 6, respectively) to enrich for mitotic cells (confirmed by enhanced expression of cyclin B1).Western blotting demonstrated the expression of the indicated proteins.(B) The extracts in panel A were immunoprecipitated with an acetyl-lysine antibody (lanes 2-7) or an HA control antibody with HFK + HPV16 extract (lane 1).Western blotting demonstrated the level of acetyl-lysine pull-down of the indicated proteins.(C) The extracts in panel A were immunoprecipitated with a SIRT1 antibody (lanes 2-7) or an HA control antibody with HFK + HPV16 extract (lane 1).Western blotting demonstrated the level of SIRT1 pull-down of the indicated proteins.(D) RNA levels of the indicated genes were determined in DTB-arrested and mitotic-enriched cells.Significant changes are indicated with *, P value < 0.05.

Fig. 1A demonstrating
Fig.1Ademonstrating significant changes in TopBP1, SIRT1, E2, CHK2, and p53 between the samples.One of the functions of SIRT1 during mitosis is to deacetylate histones and promote chromatin condensation(31,32).The reduction in SIRT1 function during mitosis could therefore alter transcription from the viral and host genome in HFK + HPV16 cells.Figure1Ddemonstrates that there is a significant increase in E2 and p53 RNA levels in the mitotically enriched HFK + HPV16 cells; p53 RNA levels are not increased in mitosis in either N/Tert-1 Vec or HFK + E6/E7 cells.With SIRT1 there is a reduction in mitotic HFK + HPV16 cells and an increase in HFK + E6/E7, although neither reached significance.When paired, there is a significant difference between mitotic SIRT1 RNA levels in HFK + HPV16 cells versus HFK + E6/E7 cells.There is no significant change in the SIRT1 levels in N/Tert-1 Vec cells.Compared to the levels of protein changes observed (Fig.1A), the changes in RNA levels were minimal although may contribute to the changes in protein levels observed in mitosis (Fig.1A; Fig.S1).There is no significant change in TopBP1 or CHK2 RNA levels in any of the samples.As the changes in p53 and SIRT1 levels were not observed in HFK + E6/E7 (Fig.1), and E2 increases TopBP1 protein levels during mitosis(20,21), the ability of E2 to regulate SIRT1 function during mitosis via interaction with TopBP1 was investigated in N/Tert-1 and U2OS cells (Fig.2).Figure2Ademonstrates that in N/Tert-1 cells stably expressing E2-WT, there is an increase in TopBP1, E2, CHK2, and p53 during mitosis, which is similar to the HFK + HPV16 cells (Fig.1A).An E2 mutant that cannot bind TopBP1 (E2-S23A), did not increase the expression of TopBP1, p53, CHK2, or E2 during mitosis (compare lane 6 with lane 4).Ac-p53 (K382) expression was increased in mitosis in the E2-WT cells; this lysine is targeted by SIRT1 and confirms SIRT1 inactivation during mitosis.These data were generated from two different gels, the separation of which is indicated by the lane number position.An acetyl lysine immunoprecipitation demonstrated increased acetylation of TopBP1, CHK2 and E2 during mitosis, and a slight increase in acetylated p53 (compare lane 5 with lanes 3 and 7).In both N/Tert-1 Vec and E2-S23A cells, there is a reduction in p53 protein levels during mitosis (Fig.2A).Unlike HFK + HPV16 cells, E2-WT did not reduce SIRT1 protein levels in mitosis when compared with non-mitotic cells (Fig.2A, lanes 3 and 4), although SIRT1 protein levels did not increase as they do in Vec and E2-S23A cells (lanes 2 and 6, Fig.2A).However, as in HFK + HPV16 cells, there is a reduction in SIRT1 acetylation levels during mitosis only in the presence of E2-WT (Fig.2B, lane 5).Figure2Cdemonstrates that the ability of SIRT1 to complex with p53, CHK2, and E2 during mitosis is abrogated only in E2-WT cells, as it is in HFK + HPV16 cells.FigureS2quantitates repeats of Fig.2Ademonstrating significant changes in TopBP1, SIRT1, E2, and p53 between the samples.Figure2D-2F established that, in U2OS cells, E2-WT has the same phenotype as in N/ Tert-1 cells (repeats of Fig.2Dare quantitated in Fig.S3).E2, TopBP1, SIRT1, and p53 RNA levels were not significantly different during mitosis in the Vec, E2-WT, and E2-S23A N/ Tert-1 (Fig.S4) or U2OS (Fig.S5) cells when compared with non-mitotic cells.The results in Fig.2demonstrate that E2 manipulates SIRT1 function during mitosis in a TopBP1 interaction-dependent manner, and Fig.1confirms this happens in HFK + HPV16 cells.E2 interaction with TopBP1 is required for increased p53 acetylation on lysine 382, an acetylation known to stabilize p53.We next determined the E2 residues that regulate E2 acetylation and stability during mitosis.

FIG 2
FIG 2 An E2-TopBP1 interaction is required to turn-off SIRT1 function and increase acetylation during mitosis.(A) N/Tert-1 Vec (pcDNA vector control) (lanes 1 and 2), N/Tert-1 E2-WT (lanes 3 and 4), and N/Tert-1 E2-S23A (TopBP1 binding interacting mutant, lanes 5 and 6) were DTB (lanes 1, 3, and 5) and released for 16 h to enrich for mitotic cells (lanes 2, 4, and 6).Western blotting demonstrated the expression of the indicated proteins.Please note that the results are presented from two different blots, separated by the lane numbers.Therefore, there are two GAPDH control lanes.(B) The extracts in panel A were immunoprecipitated with an acetyl-lysine antibody (lanes 2-6) or an HA control antibody with E2-WT extracts (lane 1).Western blotting demonstrated the level of acetyl-lysine pull-down of the indicated proteins.(C) The extracts in panel A were immunoprecipitated with a SIRT1 antibody (lanes 2-7) or an HA control antibody with HFK + HPV16 extract (lane 1).Western blotting demonstrated the level of SIRT1 pull-down of the indicated proteins.Panels D-F represent a repeat of panels A-C in U2OS cells.

FIG 4
FIG 4 Aberrant interaction of E2 lysine mutants with mitotic chromatin.(A) U2OS cells stably expressing the indicated E2 proteins were generated.Western blotting demonstrated the expression of the indicated proteins.(B) Cells were grown and fixed without enrichment for mitotic cells.Random mitotic cells are indicated with white arrows and E2 (green) and TopBP1 (red) staining carried out.(C) The number of mitotic bodies that retained E2 or TopBP1 expression was determined using a Keyence imaging system.(D) The Keyence system also determined the nuclear/cytosolic localization of the indicated E2 proteins.(E) The E2 K111Q mutant complexed with mitotic chromatin but removed TopBP1 generating chromatin bridges (quantitated in panel F).Significant changes are indicated with *, P value < 0.05.

FIG 7
FIG 7 The E2-TopBP1 interaction induces a global mitotic activation of the DDR.(A and B) The indicated protein extracts were western blotted with pS-T/Q (top panels) and GAPDH (bottom panels).

FIG 8 A
FIG 8 A model explaining the regulation of E2 stability during mitosis.See text for details.

TABLE 1
Mutation of E2 lysine 111 or 112 abrogates the immortalization potential of HPV16 a